Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments.

Funatsu, Takashi; Kono, Eiji; Higuchi, Hideo; Kimura, Sumiko; Ishiwata, Shin’ichi; Yoshioka, Toshitada; Maruyama, Kosçak; Tsukita, Shöichiro

doi:10.1083/jcb.120.3.711

Cited by 115 publications

(98 citation statements)

References 50 publications

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“…1 mm) from Z-disc to M-line [15]. Each titin molecule (figure 1a) is associated with one thick filament in the A-band and one thin filament in the I-band [23]. In each half-sarcomere, each thick filament surrounds a core of six titin filaments, and each thin filament interacts with three titin filaments that radiate from neighbouring thick filaments [24].…”

Section: Layout Of Titin Within Muscle Sarcomeresmentioning

confidence: 99%

“…Because titin is bound to thick filaments in the A-band and to thin filaments in the Z-disc [23], rotation of thin filaments by the cross-bridges must lead inevitably to winding of titin upon them. Rotation of the thin filaments by the cross-bridges would also produce a torque in a-actinin in the Z-disc.…”

Section: The Winding Filament Hypothesismentioning

confidence: 99%

“…The model is based on a sarcomere structure similar to rabbit psoas muscle [14,60], with a thin filament diameter of 10 nm [61] and a titin filament diameter of 4 nm [23,62]. The length of the PEVK segment is approximately 104 nm at SL ¼ 2.6 mm [14].…”

Section: Titin Winding During Isometric Force Development and Active mentioning

confidence: 99%

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Is titin a ‘winding filament’? A new twist on muscle contraction

et al. 2011

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Recent studies have demonstrated a role for the elastic protein titin in active muscle, but the mechanisms by which titin plays this role remain to be elucidated. In active muscle, Ca 2þ -binding has been shown to increase titin stiffness, but the observed increase is too small to explain the increased stiffness of parallel elastic elements upon muscle activation. We propose a 'winding filament' mechanism for titin's role in active muscle. First, we hypothesize that Ca 2þ -dependent binding of titin's N2A region to thin filaments increases titin stiffness by preventing low-force straightening of proximal immunoglobulin domains that occurs during passive stretch. This mechanism explains the difference in length dependence of force between skeletal myofibrils and cardiac myocytes. Second, we hypothesize that cross-bridges serve not only as motors that pull thin filaments towards the M-line, but also as rotors that wind titin on the thin filaments, storing elastic potential energy in PEVK during force development and active stretch. Energy stored during force development can be recovered during active shortening. The winding filament hypothesis accounts for force enhancement during stretch and force depression during shortening, and provides testable predictions that will encourage new directions for research on mechanisms of muscle contraction.

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Section: Layout Of Titin Within Muscle Sarcomeresmentioning

confidence: 99%

Section: The Winding Filament Hypothesismentioning

confidence: 99%

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Is titin a ‘winding filament’? A new twist on muscle contraction

et al. 2011

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“…Connectin positions the myosin filament at the center of a sarcomere in vertebrate striated muscle myofibrils by linking the myosin filament to the Z-line [4]. The connectin molecule is a long, thin, elastic filament and is responsible for passive tension generation of stretched sarcomeres [5].…”

Section: Introductionmentioning

confidence: 99%

Binding of the N‐terminal 63 kDa portion of connectin/titin to α‐actinin as revealed by the yeast two‐hybrid system

et al. 1997

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Connectin/titin is a 3000 kDa protein which links the myosin filament to the Z-line in vertebrate striated muscle sarcomeres. To search for the Z-line proteins to which connectin binds, the yeast two-hybrid system was applied using cDNA coding the N-terminal 63 kDa fragment of connectin. Two clones coding the C-terminal half region of a-actinin (amino acids, 343-897 and 446-897) were obtained. Enzyme-linked immunosorbent assay clearly demonstrated the interactions of a-actinin and the N-terminal 63 kDa fragment of connectin in vitro. Thus it is concluded that the N-terminal 63 kDa portion of connectin binds to a-actinin in the Z-line of myofibrillar sarcomeres.

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“…Instead of nebulin, nebulette is present in cardiac preparations (Moncman and Wang 1995), but since its molecular weight is small (107 kD) and it is localized close to the Z-line, the interaction between nebulette and actin molecules may become normal with the actin/thin filament reconstitution. Connectin (titin) (Maruyama et al 1976;Wang et al 1979) does not appear to interfere with the actin/thin filament reconstitution, primarily because it wraps only around the thick filament in the A-band area, and it runs in parallel with the actin/thin filament in the I-band area, except at a region near the Z-line (Funatsu et al 1990(Funatsu et al , 1993. Connectin is depicted as the thin line in Fig.…”

Section: Reconstitution Of the Actin/thin Filament In Skeletal Musclesmentioning

confidence: 99%

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

Kawai

Ishiwata

2006

J Muscle Res Cell Motil

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The technique of selective removal of the thin filament by gelsolin in bovine cardiac muscle fibres, and reconstitution of the thin filament from isolated proteins is reviewed, and papers that used reconstituted preparations are discussed. By comparing the results obtained in the absence/presence of regulatory proteins tropomyosin (Tm) and troponin (Tn), it is concluded that the role of Tm and Tn in force generation is not only to expose the binding site of actin to myosin, but also to modify actin for better stereospecific and hydrophobic interaction with myosin. This conclusion is further supported by experiments that used a truncated Tm mutant and the temperature study of reconstituted fibres. The conclusion is consistent with the hypothesis that there are three states in the thin filament: blocked state, closed state, and open state. Tm is the major player to produce these effects, with Tn playing the role of Ca 2+ sensing and signal transmission mechanism. Experiments that changed the number of negative charges at the N-terminal finger of actin demonstrates that this part of actin is essential to promote the strong interaction between actin and myosin molecules, in addition to the well-known weak interaction that positions the myosin head at the active site of actin prior to force generation. KeywordsActin; Regulatory proteins; Tropomyosin; Troponin; Myosin; Gelsolin; Cross-bridge kinetics; Sinusoidal analysis; BDM Thin filament reconstituted muscle fibre systemFor the purpose of understanding the cross-bridge interaction with actin and the role of regulatory proteins in this interaction, we reconstituted the thin filament in cardiac muscle fibres. This method is schematically represented in Fig. 1. The thin filament is first removed by gelsolin (Fig. 1A to 1B), followed by sequential reconstitution with G-actin (Fig. 1C), and regulatory proteins, tropomyosin (Tm) and troponin (Tn) (Fig. 1D). Gelsolin is an actin and thin filament severing protein, hence its action is specific and it does not disrupt other sarcomeric structure. However, overtreatment with gelsolin would disrupt the Z-line structure, because actin is one of the main constituents in the Z-line. The thin filament reconstitution method was initially developed at Ishiwata's laboratory and the key properties of the Correspondence to: Masataka Kawai, masataka-kawai@uiowa.edu. reconstituted fibres, such as the recovery of isometric tension, the tension-pCa relationship, and the function of spontaneous oscillatory contraction, were examined on skeletal fibres (Funatsu et al. 1994), followed by cardiac fibres (Fujita et al. 1996; Ishiwata 1998, 1999; for brief review see Ishiwata et al. 1998). In these works, it was demonstrated that bovine cardiac fibres were successful as the reconstituted system, in which actin, Tm and Tn can be replaced with those prepared from other muscles, for example, rabbit skeletal muscle proteins (Fujita et al. 1996;Fujita and Ishiwata 1999). The reconstitution method in cardiac fibres was successfully applied at ...

show abstract

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments.

Cited by 115 publications

References 50 publications

Is titin a ‘winding filament’? A new twist on muscle contraction

Is titin a ‘winding filament’? A new twist on muscle contraction

Binding of the N‐terminal 63 kDa portion of connectin/titin to α‐actinin as revealed by the yeast two‐hybrid system

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

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